Lateral flow test strip assay for osteoporosis

ABSTRACT

A lateral flow test strip (LFTS) platform measures osteocalcin (OC) in saliva to identify early indications of bone loss and minimize bone fracture risk associated with osteoporosis. The OC assay embodiments are based on the experimentally identified optimal markers which exhibit selectivity with very low false positives, and sensitivity relevant to clinical requirements. A prospective clinical study sampling of 20 patients demonstrated excellent correlation of OC in saliva with bone mineral density (BMD). Salivary OC and Dpd levels were validated with a standard commercial ELISA kit against serum (OC) and urine (Dpd). Multiplexed LFTS are used to increase specificity of an assay.

RELATED APPLICATIONS

This application for patent claims the benefit of and is a continuationof U.S. non-provisional patent application Ser. No. 15/078,519, filed onMar. 23, 2016 and claims the benefit of provisional application62/136,929 filed on Mar. 23, 2015. Both applications are incorporatedherein in their entirety.

GOVERNMENT LICENSE RIGHTS

This invention was made with government support under contract#41R43DE022478-01 awarded by the National Institute of Health. Thegovernment has certain rights in the invention.

FIELD OF THE INVENTION

This field of this disclosure relates generally to the assay ofbiological markers for bone loss.

BACKGROUND

Bone health is regulated in a tightly coupled metabolic process betweenbone formation (by osteoblasts) and bone resorption (by osteoclasts). Inhealthy bone these processes are in balance; however, these rates maybecome uncoupled due to diseases affecting this regulation (Paget'sdisease, metastatic bone cancer), or hormonal changes, as inpostmenopausal women. When bone resorption occurs more than boneformation, a net loss in bone mineral density (BMD) results, which canlead to diseases such as osteoporosis. The traditional approach tomeasuring BMD is dual energy X-ray absorption (DEXA), reported as aT-score (standard deviation from mean BMD) or Z-score (standarddeviation from age-matched BMD). However, DEXA is an expensiveprocedure, and is not readily available for general populationscreening. The development of a viable screening diagnostic exam forearly detection of BMD loss is a long-standing problem.

SUMMARY

Disclosed is a system for screening is the detection of biomarkers ofbone formation and degradation, which can be assayed in human serum orurine via concentrations of osteocalcin (OC) and deoxypyridinoline(Dpd), respectively. Various embodiments of diagnostic systems disclosedreliably identify the concentrations of these biomarkers in human salivawhich provides clinicians an opportunity to improve the diagnosis andprevention of osteoporosis, as well as provide a noninvasive method forconvenient population screening.

Disclosed are embodiments of a lateral flow test strip (LFTS) platformwhich measure osteocalcin (OC) and deoxypyridinoline (Dpd) in saliva toidentify early indications of bone loss and minimize bone fracture riskassociated with osteoporosis. The OC assay embodiments are based on theexperimentally identified optimal markers which exhibit selectivity withvery low false positives, and sensitivity relevant to clinicalrequirements. A prospective clinical study sampling of 20 patientsdemonstrated excellent correlation of OC in saliva with bone mineraldensity (BMD). Salivary OC and Dpd levels were validated with a standardcommercial enzyme-linked immunosorbent assay (ELISA) kit against serum(OC) and urine (Dpd).

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows a single biomarker lateral flow test strip.

FIG. 2A shows a photograph of sample quantified lateral flow assay (LFA)test strips with measured levels of Dpd.

FIG. 2B shows a photograph of sample quantified LFA test strips withmeasured levels of OC.

FIG. 3 shows a multiplex biomarker lateral flow test strip and teststrip reader apparatus.

FIG. 4 shows a chart of clinical patient results for testing salivaryOC.

FIG. 5A shows a chart of measured salivary OC compared against measuredOC from serum.

FIG. 5B shows a chart of measured salivary Dpd compared against measuredDpd from urine.

FIG. 6 shows a correlation computation for the embodiment measured OCagainst the laboratory ELISA measured OC.

FIG. 7A shows a chart of LFTS measured OC compared with BMD levels insubjects.

FIG. 7B shows a detailed graph comparing levels of subject BMD andmeasured OC from the developed embodiment LFTS.

FIG. 8 is a chart of tested and identified optimal combinations ofreagents for detecting biomarkers.

FIG. 9 is a chart of identified biomarkers suitable for variousembodiments of the disclosed LFTS platform.

DETAILED DESCRIPTION

The disclosed LFTS platform is a rapid immunochromatographic assaycomprised of a test strip with several membranes that house all thereagents necessary for the test. The analyte of interest is applied inthe sample medium (OC or Dpd in saliva), wherein it is captured in thetest in a sandwich-antibody immunocomplex coupled to fluorescentdetection. Monoclonal antibodies specific for OC or Dpd are conjugatedto fluorescently labeled microparticles and deposited on the conjugatepad. Upon adding the sample to the sample pad, the saliva resolubilizesthe dried antibody conjugates and forms an analyte-antibody conjugatecomplex, which is captured by another monoclonal antibody specific forOC or Dpd immobilized to the nitrocellulose membrane. Excitation ofcaptured fluorescent particles generates signal response proportional tothe concentration of reagent in the sample.

Shown in FIG. 1 is an exemplar LFTS 101 for a single biomarkerconfiguration. The LFTS platform includes sample pad 102, conjugate pad103, nitrocellulose membrane 104 and wicking pad 110. Detectorantibodies (OC or Dpd) 105 are carried on the conjugate pad and captureantibodies (OC or Dpd) 108 are carried by the capture line 106. Acontrol line 109 is also located on the nitrocellulose membrane. Flow ofthe sample 107 is mediated by a combination of capillary action throughthe membranous components from the sample pad 102 to the absorbent pad110, and by pressure from the liquid in the sample pad pushing towardthe absorbent pad.

Experimental results for determining optimal configuration of the LFTSsystem were conducted by the following procedure. Separate, finalizedtest strips for OC and Dpd were placed in plastic cassettes for testing.The cassettes have an open window to view the test results, and a sampleport where the sample is applied. In this embodiment the widely-usedQiagen ESE test strip fluorescent reader was adapted and calibrated withcustomized optical settings of emission and excitation wavelengths toread the selected fluorescent label. Other readout devices can be usedfor the reported platform measurements; such as LRE/SOFIA by Quidel,Cell-phone readout by Holomics, and RDS-1500 PRO by Detekt Biomedical.Saliva samples from 20 donor patients were obtained. Collected salivasamples were kept frozen at −80° C. until tested. Frozen samples werethawed, centrifuged to remove large particulates, and diluted 1 to 1with our running buffer. Tests were performed in triplicate with eachsaliva sample. After adding 100 uL of sample volume to each test, thetests flowed for 10 minutes before the results were read with the ESEreader.

Shown in FIG. 2A and FIG. 2B are exemplar samples. Shown are sampleswith Dpd concentrations of 30 nmol/L 201A, 15 nmol/L 202A, 7.5 nmol/L203A, 3.7 nmol/L 204A, 1.8 nmol/L 205A and 0 nmol/L 206A. Formation ofsandwich antibody detection with decreasing concentrations of Dpd.Fluorescence was observed under an ultra-violet (UV) light source. Shownin FIG. 2B are exemplar OC samples with intensity observed from spikingsynthetic OC in saliva. Shown tests were run in cassettes. Fluorescencewas observed under UV light source for the samples 0 ng/mL 201B, 5 ng/mL202B, 10 ng/mL 203B, 15 ng/mL 204B and 30 ng/mL 205B

Shown in FIG. 3 is the fluorescent reader apparatus 301 utilized forsingle and multiplex test strips. Shown in the inset is an embodimentfor a multiplex LFTS 302 with multiple detection lines (multiplex) 303and 304 and control line 305.

A standard commercial enzyme-linked immunosorbent assay (ELISA) test kitwas used to validate the embodiment LFTS platform with patient salivasamples. A correlation value of 0.85 was obtained with OC.

In various embodiments salivary OC and Dpd concentrations are correlatedwith serum (OC) and urinary (Dpd) levels from the same patient usingELISA measurements. Samples are normalized by protein concentration toadjust for salivary specific gravity. The resulting high correlationconfirms the reliability of salivary markers for the disclosedembodiment.

Shown in FIG. 4 are results for patient samples categorized into threemain groups—normal, osteopenic, and osteoporotic—showed high amounts ofOC in normal patients, and low OC in osteopenic/osteoporotic patients.

Note that for purposes of testing various embodiments, after adding 100uL of sample volume to each test, the tests flowed for 10 minutes beforethe results were read with the ESE reader.

To determine the optimum LFA components, incremental assay optimizationsteps were carried out for both OC and DPD assays, including captureantibody concentration, assay running buffers, purification of theantibody reagents, addition of surfactants, selection of LFA membranes,conjugate pad selection, and fabrication/drying protocols.

FIG. 8 shows a chart of tested capture and detector reagents along withthose combinations which proved to be effective for the disclosedembodiment. To identify the antibody pair with the best affinity toosteocalcin (OC), OC reagents were screened from Fitzgerald, NovusBiologicals, Thermo Scientific, and Hytest that includes mouse anti-OCmonoclonal and rabbit polyclonal antibodies for sandwich pairing andantigen testing. The following pairs were the only combinations thatbound synthetic osteocalcin as membrane/detector: 940/939, 11F8/939,2H9/939, 940/H10, 2H9/H10, 940/3G8, 11F8/3G8, 2H9/3G8. Two pairs boundstrongly with native osteocalcin as membrane/detector: 940/H10, and2H9/H10, and were used for further assay optimization and testing. Mousemonoclonal antibodies against DPD were acquired from Quidel and screenedalong with an antibody from a human DPD ELISA kit (MyBioSource).

Salivary OC and Dpd concentrations were correlated with serum (OC) andurinary (Dpd) levels from the same patient using ELISA measurements.Samples were normalized by protein concentration to adjust for salivaryspecific gravity. The resulting high correlation suggested thereliability of salivary markers. Shown in FIG. 5A are results forsalivary experimental measurements vs. the serum OC validation marker.Shown in FIG. 5B are results for salivary experimental measurements vs.the urine Dpd validation marker.

A standard commercial ELISA test kit was used to validate the disclosedLFTS platform with patient saliva samples. A correlation value of 0.85was obtained with OC. FIG. 6 shows the correlation of OC measurements.

In various embodiments, the disclosed quantifiable LFTS may containmultiplexed biomarkers for both OC and Dpd. The LFTS shown in FIG. 3illustrates a multiplexed LFA.

In various embodiments, additional biomarkers are utilized in thedisclosed LFTS in either the disclosed single or multiplexedquantifiable methodology. Bone turnover biomarkers are represented incompounds such as collagen precursors, enzymes, and by-products, ordegradation products involved with the bone formation (osteoblast) andbone resorption (osteoclast) processes. In various embodiments, one ormore of these biomarkers are utilized in the disclosed LFTS for optimalor additional accuracy for screening examination. FIG. 9 provides achart of the biomarkers identified for use in these embodiments.

The disclosed sensitive lateral flow assay-based technique are capableof the detecting bone formation marker osteocalcin in saliva atclinically relevant levels which has been demonstrated and correlated toBMD, utilizing the disclosed readout system which as disclosed is easilyintegrated in a single platform for point-of-care (POC) applications.

Other embodiments of the invention utilize equivalent monoclonalantibody capture and detector reagents and reagent pairs for the assay.

What has been described herein is considered merely illustrative of theprinciples of this invention. Accordingly, it is well within the purviewof one skilled in the art to provide other and different embodimentswithin the spirit and scope of the invention.

What is claimed is:
 1. A lateral flow test strip for screening forosteoporosis comprising: a) a sample pad for accepting a subject samplebodily fluid; b) a migration membrane; c) a plastic backing; d) aconjugate pad deposited with a first fluorescently labeled detectorantibody; e) a first capture line comprising a first capture antibodyspecific to osteocalcin wherein a first antibody pair comprising thefirst capture antibody and the first fluorescently labeled detectorantibody are chosen from the group consisting of: i. 940/939, ii.11F8/939, iii. 2H9/939, iv. 940/H10, v. 2H9/H10, vi. 940/3G8, vii.11F8/3G8.
 2. The lateral flow test strip of claim 1 also comprising asecond capture line comprising a second capture antibody reactivespecific to: a) deoxypyridinoline, b) urinary calcium, c) tartareresistant acid phosphtase, d) bone sialoprotein, e) pryidinoline, f)N-telopeptide, g) C-telopeptide, h) C-terminal telopeptide of type Icollagen i) bone-specific alkaline phosphatase, j) pro-collagen Iextension peptides, k) amino terminal.
 3. The lateral flow test strip ofclaim 1 wherein the conjugate pad deposited with the first fluorescentlylabeled detector antibody is conjugated to fluorescent microparticlesbeing paired with the first capture antibody being immobilized on themigration membrane.
 4. The lateral flow test strip of claim 1 whereinthe bodily fluid is whole saliva.
 5. The lateral flow test strip ofclaim 1 wherein the bodily fluid is urine.
 6. The lateral flow teststrip of claim 1 also comprising a second capture line comprising asecond capture antibody specific to osteocalcin wherein a secondantibody pair comprising the second capture antibody and a secondfluorescently labeled detector antibody are chosen from the groupconsisting of: a) 940/939, b) 11F8/939, c) 2H9/939, d) 940/H10, e)2H9/H10, f) 940/3G8, g) 11F8/3G8.
 7. The lateral flow test strip ofclaim 6 wherein the first capture antibody and the second captureantibody are the same.
 8. A system for immunochromatographic bone lossmarker assay comprising: a) a lateral flow test strip comprising: i) asample pad for accepting a subject sample bodily fluid; ii) a migrationmembrane; iii) a plastic backing; iv) a conjugate pad deposited with afirst fluorescently labeled detector antibody; v) a first capture linecomprising a first capture antibody specific to osteocalcin wherein thefirst capture antibody and the first fluorescently labeled detectorantibody are chosen from the group consisting of: (1) 940/939, (2)11F8/939, (3) 2H9/939, (4) 940/H10, (5) 2H9/H10, (6) 940/3G8, (7)11F8/3G8. b) a calibrated florescence test strip reader.
 9. The systemof claim 8 wherein the conjugate pad deposited with the firstfluorescently labeled detector antibody is conjugated to fluorescentmicroparticles being paired with the first capture antibody beingimmobilized on the migration membrane.
 10. The system of claim 8 whereinthe lateral flow test strip also comprises a second capture linecomprising a second antibody capture antibody reactive specific to: a)deoxypyridinoline, b) urinary calcium, c) tartare resistant acidphosphtase, d) bone sialoprotein, e) pryidinoline, f) N-telopeptide, g)C-telopeptide, h) C-terminal telopeptide of type I collagen i)bone-specific alkaline phosphatase, j) pro-collagen I extensionpeptides, k) amino terminal.
 11. The system of claim 8 wherein thebodily fluid is whole saliva.
 12. The system of claim 8 wherein thebodily fluid is urine.
 13. The system of claim 8 wherein theimmunochromatographic assay also comprises a diagnostically correlatedevaluation for reader output measurements of reagent lines following theapplication of a serum specimen to the lateral flow test strip.
 14. Amulti-plexed lateral flow test strip for screening for osteoporosiscomprising: a) a sample pad for accepting a subject sample bodily fluid;b) a migration membrane; c) a plastic backing; d) a conjugate paddeposited with a first fluorescently labeled detector antibody; e) afirst capture line comprising a first capture antibody specific toosteocalcin wherein a first antibody pair comprising the first captureantibody and the first fluorescently labeled detector antibody arechosen from the group consisting of: f) a second capture line comprisinga second capture antibody specific to secondary marker for osteocalcinwherein a second antibody pair comprise a second capture antibody and asecond fluorescently labeled detector antibody. are chosen from thegroup consisting of i. osteocalcin, ii. deoxypyridinoline, iii. urinarycalcium, iv. tartare resistant acid phosphtase, v. bone sialoprotein,vi. pryidinoline, vii. N-telopeptide, viii. C-telopeptide, ix.C-terminal telopeptide of type I collagen x. bone-specific alkalinephosphatase, xi. pro-collagen I extension peptides, xii. amino terminal.